CN105779568B - The kit and its application method that sperm quality is assessed after a kind of In-vitro Capacitation - Google Patents
The kit and its application method that sperm quality is assessed after a kind of In-vitro Capacitation Download PDFInfo
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- CN105779568B CN105779568B CN201610147270.4A CN201610147270A CN105779568B CN 105779568 B CN105779568 B CN 105779568B CN 201610147270 A CN201610147270 A CN 201610147270A CN 105779568 B CN105779568 B CN 105779568B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
Abstract
The kit and its application method assessed the present invention provides sperm quality after a kind of In-vitro Capacitation.The kit includes detection buffer 1, BWW culture solution, In-vitro Capacitation liquid, fluorescent reagent, sialidase standard stock solution and detection buffer 2；The detection buffer 1 is phosphate buffer or BWW culture solution；The detection buffer 2 is the phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin(BSA).By after sperm microcytotoxicity in capacitation liquid sialidase activity detection, for clinical reason, unknown male sterility patient provides diagnosis basis and clinical consultation.
Description
Technical field
The invention belongs to medical detection technologies, and in particular to the kit that sperm quality is assessed after a kind of In-vitro Capacitation
And its application method.
Background technique
With the continuous development of social industry, the total incidence of infertile couples is significantly raised.European genesiology meeting in 2004
Statistics: in the couple at child-bearing age 1 year cannot the person of pregnancy account for 25%, wherein 15% seeks to treat, bridegroom's or husband's side factor causes infertility to account for 50%.Male
The cause of disease sexual disorders (1.7%) of infertility, varicocele (12. 3%), reproductive tract infection (6.6%), congenital hair
Exception (2.1%) acquired disease day after tomorrow (2.6%) is educated, endocrine disturbance (0.6%) is immunized sexual factor (3.1%), other exceptions
(3%), it but is up to 60% ~ 75% patient and can not find reason, referred to as idiopathic male infertility, they only show as few essence, weak essence
Or the sperm qualities such as teratozoospermia are abnormal.The male sterility of unknown cause may be caused due to many factors, such as long-term stress
Environmental factor causes endocrine disturbance, active oxygen and gene defect etc..
For many years, traditional semen routine analysis is always the most basic clinical indices for being used to judge male fertility.Face
The real sterile reason of the male sterility patient of the overwhelming majority is still unclear on bed.Especially clinically about three/
One infertile patients, the Sperm routine analysis result of male are normal and close to normally.This kind of patient is clinically divided into unknown original
Because of sterility.Therefore, for a long time, there is very big difficulty in the clinical diagnosis to male sterility.It is most important the reason is that conventional
Semen analysis only measures the quantity of sperm, viability, activity ratio and form.These indexs can only reflect most basic sperm matter
Amount, cannot reflect all sperm functions, for example, the maturation and DNA damage of sperm nucleus, sperm and human oocyte zona pellucida association reaction
With penetrate, acrosome situation and reaction and with vitellinae membrana binding ability etc..Therefore, it is necessary to establish special sperm function tests ability
Measure the above function.Capacitation is the necessary condition that sperm ovum binding forms fertilized eggs, although the correlation of capacitation is ground
Study carefully and have some progress, but still there are problems that much not yet illustrating.
In clinical position, a part of primary or secondary Infertility male patient utilizes existing conventional semen quality check item
Mesh prompts sperm and semen quality without exception.Currently used sperm detection means is CASA (computer aided sperm analysis
Computer aided of semen analysis) and AsAb etc..Current the most widely used CASA technology
With some morphologic coherent detections, the mobility and sperm ratio living of sperm can be mainly evaluated, to speculate that it enters female
The fertility in sexual reproduction road, and rely on current sperm function appraisement system, to the evaluation of sperm quality and function, there are one
Fixed defect, and be difficult to provide corresponding clinical consultation to patient.In fact, this is only a side for evaluating sperm function
Face, we are specifically contemplated that the factor of the sperm fecundity that may also have an impact not being revealed more.
First patent 201310431847.0 proposes a kind of sialidase detection method for influencing sperm function.First
Step intuitively quantifies expression of the sialidase in sperm by monosperm Fluorescence Intensity Assays, or utilizes glimmering under microscope
Expression of the intuitive observation sialidase of light imaging in sperm；If sialidase high expression in sperm, carries out second step,
Sperm is set to influence the sialidase detection method capacitation of sperm function using In-vitro Capacitation liquid, and with Fluorometric assay sialidase
Activity.Although the patent discloses the detection of expression and primary activity of the sialidase protein molecule in sperm.But in method
Include the detection of expression of sperm sialidase NEU1/3, needs fluorescence microscope or flow cytometer, have the disadvantage in that 1)
Techniqueflow is complicated；2) experimental facilities and operator's skills and experience are required high；3) it is unsuitable for being applied to clinical labororatory
It is used with those having ordinary skill in the art；4) kit of abcam company is used, it is expensive.In addition, due to the sperm count of different samples
Amount is different, and the protein value of expression is different, and corresponding enzyme activity value is also different, and therefore, the enzyme activity value of more different samples is not merely
Science.
Summary of the invention
In view of the above technical problems, the present invention provides after a kind of In-vitro Capacitation sperm quality assess kit and its make
Use method.By after sperm microcytotoxicity in capacitation liquid sialidase activity detection, assess capacitation after sperm quality, to face
Bed reason unknown male sterility patient provide diagnosis basis and clinical consultation.
In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme that:
The kit that sperm quality is assessed after a kind of In-vitro Capacitation, it is characterised in that: the kit includes that detection is slow
Fliud flushing 1, BWW culture solution, In-vitro Capacitation liquid, fluorescent reagent, sialidase standard stock solution and detection buffer 2.
The detection buffer 1 is PBS(phosphate buffer) or BWW culture solution.
The detection buffer 2 is the PBS containing 0.05 mmoL/L sodium acetate and 0.25 μ g/ μ l bovine serum albumin(BSA).Inspection
The buffer system for surveying buffer 2 is to detect specific preparation for sialidase activity.
Preferably, the pH value of the sodium acetate is 5-6, is for the optimal working environment of saliva enzymatic activity.
The In-vitro Capacitation liquid is the HTF(human tubal fluid containing HSA (human serum albumins) 5mg/ml).
The sialidase standard stock solution is C.perfringens sialidase (AUS), and concentration 5U/ml can be convenient
Be diluted to these concentration gradients of 5/2.5/1.25/0.625/0.312/0 mU/ml.It is suitable using C.perfringens sialidase
Detection method for a small amount of sperm of the invention.
The fluorescent reagent is 2 ' -4-methyl umbelliferone base-α-D-N- n acetylneuraminic acid n sodium, with higher sensitive
Degree.
The specific structure of the 2 ' -4-methyl umbelliferone base-α-D-N- n acetylneuraminic acid n sodium is as follows:
Preferably, the concentration of the fluorescent reagent is 10mM, provides the concentrate of fluorescent reagent, it is ensured that its stability,
It is not easily decomposed and is quenched, can separately reduce volume, be convenient for mounted box.
The application method for the kit that sperm quality is assessed after In-vitro Capacitation of the present invention: specific step is as follows:
A, the washing of sperm
Collect fresh liquefaction semen sample, detection buffer 1 washs sperm, sufficiently remove remaining refining ingredient, then from
The heart isolates sperm.
Detection buffer 1 is first added into semen sample and washs by the present invention, then is centrifugally separating to obtain Sperm pellets.This
It is since refining itself is sticky, being directly centrifuged to it cannot be such that all sperms in sperm sufficiently precipitate, and it is right that detection buffer 1 is added
After it is diluted, centrifugally operated effect is more preferable.
Preferably, 3 centrifugations are carried out to sperm, guarantees in the case where detecting no refining interference, reduces centrifugation as far as possible
The number of sperm guarantees sperm motility.
B, the In-vitro Capacitation of sperm
BWW culture solution is added into the sperm that step A is isolated, using upper reaches method, in 5% CO2It is trained in cell incubator
It supports, harvests upstream sperm, count sperm quantity, the CO with In-vitro Capacitation liquid 5%23 ~ 4 h of sperm is incubated in cell incubator；
Centrifuge separation, the supernatant collected are centrifugated again, collect supernatant as sample to be tested.It is preferable that vigor is obtained in upper reaches method
Sperm after, capacitation processing is carried out to it at once, experimental result is more scientific objective.
Preferably, control upstream sperm quantity is in 1X107More than, it is set according to the needs of detection sensitivity.
C. in capacitation liquid sialidase activity measurement
(1) sialidase standard stock solution is pressed 5/2.5/1.25/0.625/0.312/0 mU/ml's with detection buffer 2
Concentration gradient is diluted to sialidase standard solution, and each sialidase standard solution and to be measured is sequentially added into detection plate
Sample.
The gradient of standard solution is the amount and the method for the present invention being discharged into capacitation liquid according to sperm sialidase after capacitation
The sensitivity that can be detected and set.
Preferably, the detection plate is 96 hole blackboards.The present invention measures the activity of sialidase using fluorescence method, black
The detection plate of color be to sensitivity and specificity it is optimal, can protect fluorescent dye and be not quenched by light, thus make detection
Sensitivity greatly improves.
(2) fluorescent reagent is diluted with detection buffer 2, is added into above-mentioned each sialidase standard solution and sample to be tested
Hole in, then will test plate and be placed in 37 DEG C, after being protected from light incubation 1-2 hour, with each hole fluorescence intensity of microplate reader reading, according to saliva
Phytase activity standard curve calculates the sialidase activity of sample to be tested, finally obtains sialidase activity/sperm number
Value.
Preferably, being diluted to the concentration of fluorescent reagent with detection buffer 2 is 0.05mM, is optimal working concentration.
Preferably, microplate reader reads each Kong Ying in the case where excitation wavelength and launch wavelength are respectively the wavelength of 365nm and 450nm
Luminous intensity.365nm is maximum excitation wavelength, and 450nm is best launch wavelength.
It preferably, should be within the scope of 0.3-5mU/ml, if exceeding for sample sialidase activity in the hole of detection
The sialidase activity value of sample to be tested in hole should be adjusted within this range of linearity.
The beneficial effects of the present invention are:
1, the reagent in kit of the present invention selects routine test reagent, and combines the particularity detected to sperm, drop
Low experimental cost, required instrument and equipment, consumptive material type are few, only centrifuge, microplate reader, EP pipe, pipette tips and detection plate etc.,
It is Routine Test Lab configuration；And operating method is simple, be only centrifuged, be loaded etc., there is no need to repeatedly practice obtaining
The case where operation skill, can be widely applied to clinical labororatory and those having ordinary skill in the art uses.
2, kit of the invention is used to assess the quality of sperm after capacitation, according to sialidase in unit sperm quantity
Activity value makes the critical value that crowd expresses sialidase activity, can understand clinically by unit enzyme activity value deeper time
Sperm function can be applied to reproduction auxiliary such as test-tube baby's technology.
3, since the sperm quantity of different samples is different, the protein value of expression is different, and corresponding enzyme activity value is also different, in this way
One, the enzyme activity value of more different samples is not scientific merely, if because the sperm number of a sample is seldom, but sperm matter
Amount is very high, and there are many sperm number of another sample, but sperm quality is lower, if comparing enzyme activity value merely, the former very may be used
It can be defeated by the latter, but actual conditions are, the former is higher than the latter at quality.Therefore, detection of the invention be sialidase activity/
The unit enzyme activity value of sperm number, testing result are more objective, accurate and reliable.
4, the present invention adequately washs sperm, sufficiently removes remaining refining ingredient, makes remaining egg in refining
Bletilla sialidase will not influence the accuracy of testing result；It is dense to optimize the standard items albumen being arranged when surveying sperm protein concentration
The standard items enzyme activity gradient being arranged when degree gradient and survey enzyme activity can influence the sensitive of result if gradient setting is unreasonable very much
Degree and accuracy；Enzyme activity is surveyed using black detection plate, can obviously reduce the negative effect that fluorescent reagent is quenched in light, thus greatly
Big stability, sensitivity and the accuracy for improving experimental result；Final testing result is designed as sialic acid enzyme activity/sperm number
Value, the difference between different samples can be reduced in this way, make the stability for guaranteeing testing result with more comparativity each other and can
By property.
Detailed description of the invention
Fig. 1 is the sialic acid that kit of the present invention measures non-capacitated sperm and capacitated sperm (A is mouse, and B is people) respectively
Enzymatic activity.
Specific embodiment
Property content is described in further detail for the essence of the present invention With reference to embodiment.
Embodiment 1
The kit that sperm quality is assessed after a kind of In-vitro Capacitation, the kit include detection buffer 1, BWW training
Nutrient solution, In-vitro Capacitation liquid, fluorescent reagent, sialidase standard stock solution and detection buffer 2；The detection buffer 1 is phosphorus
Acid buffer；The detection buffer 2 is the phosphoric acid containing 0.05 mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin(BSA)
Buffer.
Kit of the present invention measures the sialic acid enzyme activity of non-capacitated sperm and capacitated sperm (A is mouse, and B is people) respectively
Property, as the result is shown after capacitation the releasable sialidase of sperm into capacitation liquid.
Embodiment 2
The kit that sperm quality is assessed after a kind of In-vitro Capacitation, the kit include detection buffer 1, BWW training
Nutrient solution, In-vitro Capacitation liquid, fluorescent reagent, sialidase standard stock solution and detection buffer 2；The detection buffer 1 is BWW
Culture solution；The detection buffer 2 is slow for the phosphoric acid containing 0.05 mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin(BSA)
Fliud flushing.
The In-vitro Capacitation liquid is the HTF of the 5mg/ml containing HSA.
Embodiment 3
The kit that sperm quality is assessed after a kind of In-vitro Capacitation, the kit include detection buffer 1, BWW training
Nutrient solution, In-vitro Capacitation liquid, fluorescent reagent, sialidase standard stock solution and detection buffer 2；The detection buffer 1 is phosphorus
Acid buffer；The detection buffer 2 is the phosphoric acid containing 0.05 mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin(BSA)
Buffer.
The In-vitro Capacitation liquid is the HTF of the 5mg/ml containing HSA.
The sialidase standard stock solution is C.perfringens sialidase, concentration 5U/ml.
Embodiment 4
The kit that sperm quality is assessed after a kind of In-vitro Capacitation, the kit include detection buffer 1, BWW training
Nutrient solution, In-vitro Capacitation liquid, fluorescent reagent, sialidase standard stock solution and detection buffer 2；The detection buffer 1 is phosphorus
Acid buffer；The detection buffer 2 is the phosphoric acid containing 0.05 mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin(BSA)
Buffer.
The In-vitro Capacitation liquid is the HTF of the 5mg/ml containing HSA.
The sialidase standard stock solution is C.perfringens sialidase, concentration 5U/ml.
The fluorescent reagent is 2 ' -4-methyl umbelliferone base-α-D-N- n acetylneuraminic acid n sodium.
The pH value of the sodium acetate is 5.5.
Embodiment 5
The kit that sperm quality is assessed after a kind of In-vitro Capacitation, the kit include detection buffer 1, BWW training
Nutrient solution, In-vitro Capacitation liquid, fluorescent reagent, sialidase standard stock solution and detection buffer 2；The detection buffer 1 is phosphorus
Acid buffer；The detection buffer 2 is the phosphoric acid containing 0.05 mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin(BSA)
Buffer.
The In-vitro Capacitation liquid is the HTF of the 5mg/ml containing HSA.
The sialidase standard stock solution is C.perfringens sialidase, concentration 5U/ml.
The fluorescent reagent is 2 ' -4-methyl umbelliferone base-α-D-N- n acetylneuraminic acid n sodium.
The concentration of the fluorescent reagent is 10mM.
The pH value of the sodium acetate is 5.
Embodiment 6
The kit that sperm quality is assessed after a kind of In-vitro Capacitation, the kit include detection buffer 1, BWW training
Nutrient solution, In-vitro Capacitation liquid, fluorescent reagent, sialidase standard stock solution and detection buffer 2；The detection buffer 1 is phosphorus
Acid buffer；The detection buffer 2 is the phosphoric acid containing 0.05 mmol/l sodium acetate and 0.25 μ g/ μ l bovine serum albumin(BSA)
Buffer.
The In-vitro Capacitation liquid is the HTF of the 5mg/ml containing HSA.
The sialidase standard stock solution is C.perfringens sialidase, concentration 5U/ml.
The fluorescent reagent is 2 ' -4-methyl umbelliferone base-α-D-N- n acetylneuraminic acid n sodium.
The concentration of the fluorescent reagent is 10mM.
The pH value of the sodium acetate is 6.
Embodiment 7
The application method for the kit that sperm quality is assessed after In-vitro Capacitation of the present invention, the specific steps are as follows:
A, the washing of sperm
Collect fresh liquefaction semen sample, detection buffer 1 washs sperm, sufficiently remove remaining refining ingredient, then from
The heart isolates sperm；
B, the In-vitro Capacitation of sperm
BWW culture solution is added into the sperm that step A is isolated, using upper reaches method, in 5% CO2It is trained in cell incubator
It supports, harvests upstream sperm, count sperm quantity, the CO with In-vitro Capacitation liquid 5%23 ~ 4 h of sperm is incubated in cell incubator；
Centrifuge separation, the supernatant collected are centrifugated again, collect supernatant as sample to be tested；
C, in capacitation liquid sialidase activity measurement
(1) sialidase standard stock solution is pressed 5/2.5/1.25/0.625/0.312/0 mU/ml's with detection buffer 2
Concentration gradient is diluted to sialidase standard solution, and each sialidase standard solution and to be measured is sequentially added into detection plate
Sample.
(2) fluorescent reagent is diluted with detection buffer 2, is added into above-mentioned each sialidase standard solution and sample to be tested
Hole in, then will test plate and be placed in 37 DEG C, after being protected from light incubation 1-2 hour, with each hole fluorescence intensity of microplate reader reading, according to saliva
Phytase activity standard curve calculates the sialidase activity of sample to be tested, finally obtains sialidase activity/sperm number
Value.
Embodiment 8
The application method for the kit that sperm quality is assessed after In-vitro Capacitation of the present invention, the specific steps are as follows:
A, the washing of sperm
Collect fresh liquefaction semen sample, detection buffer 1 washs sperm, sufficiently remove remaining refining ingredient, then from
The heart isolates sperm；
B, the In-vitro Capacitation of sperm
BWW culture solution is added into the sperm that step A is isolated, using upper reaches method, in 5% CO2It is trained in cell incubator
It supports, harvests upstream sperm, count sperm quantity, the CO with In-vitro Capacitation liquid 5%23 ~ 4 h of sperm is incubated in cell incubator；
Centrifuge separation, the supernatant collected are centrifugated again, collect supernatant as sample to be tested；
C, in capacitation liquid sialidase activity measurement
(1) sialidase standard stock solution is pressed 5/2.5/1.25/0.625/0.312/0 mU/ml's with detection buffer 2
Concentration gradient is diluted to sialidase standard solution, and each sialidase standard solution and to be measured is sequentially added into detection plate
Sample.
(2) fluorescent reagent is diluted with detection buffer 2, is added into above-mentioned each sialidase standard solution and sample to be tested
Hole in, then will test plate and be placed in 37 DEG C, after being protected from light incubation 1 hour, each hole fluorescence intensity is read with microplate reader, according to sialic acid
Enzymatic activity standard curve calculates the sialidase activity of sample to be tested, finally obtains sialidase activity/sperm number value.
The detection plate is 96 hole blackboards.
Embodiment 9
The application method for the kit that sperm quality is assessed after In-vitro Capacitation of the present invention, the specific steps are as follows:
A, the washing of sperm
Collect fresh liquefaction semen sample, detection buffer 1 washs sperm, sufficiently remove remaining refining ingredient, then from
The heart isolates sperm；
B, the In-vitro Capacitation of sperm
BWW culture solution is added into the sperm that step A is isolated, using upper reaches method, in 5% CO2It is trained in cell incubator
It supports, harvests upstream sperm, count sperm quantity, the CO with In-vitro Capacitation liquid 5%23 ~ 4 h of sperm is incubated in cell incubator；
Centrifuge separation, the supernatant collected are centrifugated again, collect supernatant as sample to be tested；
C, in capacitation liquid sialidase activity measurement
(1) sialidase standard stock solution is pressed 5/2.5/1.25/0.625/0.312/0 mU/ml's with detection buffer 2
Concentration gradient is diluted to sialidase standard solution, and each sialidase standard solution and to be measured is sequentially added into detection plate
Sample.
(2) being diluted to the concentration of fluorescent reagent with detection buffer 2 is 0.05mM., it is added into above-mentioned each sialidase
In the hole of standard solution and sample to be tested, then it will test plate and be placed in 37 DEG C, after being protected from light incubation 2 hours, microplate reader is in excitation wavelength
It is respectively to read each hole fluorescence intensity under the wavelength of 365nm and 450nm with launch wavelength.According to sialidase activity standard curve
The sialidase activity of sample to be tested is calculated, finally obtains sialidase activity/sperm number value.
The detection plate is 96 hole blackboards.
Embodiment 10
The application method for the kit that sperm quality is assessed after In-vitro Capacitation of the present invention, the specific steps are as follows:
A, the washing of sperm
Collect fresh liquefaction semen sample, detection buffer 1 washs sperm, sufficiently remove remaining refining ingredient, then from
The heart isolates sperm；
B, the In-vitro Capacitation of sperm
BWW culture solution is added into the sperm that step A is isolated, using upper reaches method, in 5% CO2It is trained in cell incubator
It supports, harvests upstream sperm, count sperm quantity, the CO with In-vitro Capacitation liquid 5%23 ~ 4 h of sperm is incubated in cell incubator；
Centrifuge separation, the supernatant collected are centrifugated again, collect supernatant as sample to be tested；
C, in capacitation liquid sialidase activity measurement
(1) sialidase standard stock solution is pressed 5/2.5/1.25/0.625/0.312/0 mU/ml's with detection buffer 2
Concentration gradient is diluted to sialidase standard solution, and each sialidase standard solution and to be measured is sequentially added into detection plate
Sample.
(2) being diluted to the concentration of fluorescent reagent with detection buffer 2 is 0.05mM, is added into above-mentioned each sialidase mark
In the hole of quasi- solution and sample to be tested, then it will test plate and be placed in 37 DEG C, after being protected from light incubation 1.5 hours, microplate reader is in excitation wavelength
It is respectively to read each hole fluorescence intensity under the wavelength of 365nm and 450nm with launch wavelength.According to sialidase activity standard curve
The sialidase activity of sample to be tested is calculated, finally obtains sialidase activity/sperm number value.
The detection plate is 96 hole blackboards.
Embodiment 11
Consumptive material (sterilizes): 15mlEP pipe, 1.5mlEP pipe, 3 kinds of specification pipette tips (1ml/200 μ l/10 μ l), 96 holes are transparent
Plate, 96 hole blackboards
Instrument: high speed low temperature centrifugal machine, microplate reader
The application method for the kit that sperm quality is assessed after In-vitro Capacitation of the present invention, the specific steps are as follows:
A, the washing of sperm
1. collecting fresh liquefaction semen sample 4-5ml(to be adjusted according to patient's sperm quantity, save and transport on ice)
2. isometric PBS is added, piping and druming is uniform.
3. low-speed centrifugal (1500g/5min), discards supernatant liquid.
4. PBS to 3-4ml is added, piping and druming is uniform.
5. low-speed centrifugal (1500g5min), discards supernatant liquid, supernatant is remained to 300 μ l or so, piping and druming is uniform.
6. PBS to 1ml is added, high speed centrifugation (4 degree, 12000rpm/5min) discards supernatant liquid as far as possible.
B, the In-vitro Capacitation of sperm
1. BWW is added into Sperm pellets, upper reaches method cultivates 30min in 37 DEG C of 5%CO2 cell incubators, in harvest
Sperm is swum, counting sperm quantity (needs 1X107More than), low-speed centrifugal (1500g/5min) obtains Sperm pellets.
2. In-vitro Capacitation liquid is added, it is incubated for 4 hours in 37 DEG C of 5%CO2 cell incubators.
3. low-speed centrifugal (500g/5min), collects supernatant.
4. low-speed centrifugal (5000g/15min) again, collects supernatant.
C. in capacitation liquid sialidase activity measurement
1. with detection buffer 2 by sialidase standard stock solution by 5/2.5/1.25/0.625/0.312/0 mU/ml's
Concentration gradient is diluted to sialidase standard solution, sequentially adds 50 each sialidases of μ l into 96 hole detection plates (black)
Standard solution.
2. being 0.05mM with the concentration of fluorescent reagent is diluted to detection buffer 2, it is added into above-mentioned each hole, 50
Microlitre/hole.
3. 96 hole detection plates (black) are placed in 37 DEG C, be protected from light incubation 1-2 hour, then use microplate reader excitation wavelength/
Launch wavelength is to read each hole fluorescence intensity under 365/450nm wavelength, is calculated according to sialidase activity standard curve to be measured
The sialidase activity of sample.
4. unit enzyme activity value (U AUS/10 is calculated in the sialidase activity/sperm number that will finally obtain4
sperm)。
Should be within the scope of 0.3-5mU/ml for sample sialidase activity in the hole of detection, it should will be in hole if exceeding
The sialidase activity value of sample to be tested adjusts within this range of linearity.
Claims (9)
1. the kit of sialidase in a kind of detection external sperm capacitation liquid, it is characterised in that: the kit is by detecting
Buffer 1, BWW culture solution, In-vitro Capacitation liquid, fluorescent reagent, sialidase standard stock solution and detection buffer 2 form；It is described
Detection buffer 1 be phosphate buffer or BWW culture solution；The detection buffer 2 is containing 0.05 mmol/l acetic acid
The phosphate buffer of sodium and 0.25 μ g/ μ l bovine serum albumin(BSA)；The In-vitro Capacitation liquid is the HTF of the 5mg/ml containing HSA.
2. the kit of sialidase in a kind of detection external sperm capacitation liquid according to claim 1, it is characterised in that:
The sialidase standard stock solution is C.perfringens sialidase, concentration 5U/ml.
3. the kit of sialidase in a kind of detection external sperm capacitation liquid according to claim 1, it is characterised in that:
The fluorescent reagent is 2 ' -4-methyl umbelliferone base-α-D-N- n acetylneuraminic acid n sodium.
4. the kit of sialidase in a kind of detection external sperm capacitation liquid according to claim 3, it is characterised in that:
The concentration of the fluorescent reagent is 10mM.
5. the kit of sialidase in a kind of detection external sperm capacitation liquid according to claim 1, it is characterised in that:
The pH value of the sodium acetate is 5-6.
6. the application method of the kit of sialidase in a kind of detection external sperm capacitation liquid according to claim 1,
It is characterized by: specific step is as follows:
A, the washing of sperm
Collect fresh liquefaction semen sample, detection buffer 1 washs sperm, sufficiently removes remaining refining ingredient, then be centrifuged point
Separate out sperm；
B, the In-vitro Capacitation of sperm
BWW culture solution is added into the sperm that step A is isolated, using upper reaches method, in 5% CO2It cultivates, receives in cell incubator
Upstream sperm is obtained, sperm quantity, the CO with In-vitro Capacitation liquid 5% are counted23 ~ 4 h of sperm is incubated in cell incubator；Centrifugation point
From the supernatant collected is centrifugated again, collects supernatant as sample to be tested；
C, in capacitation liquid sialidase activity measurement
(1) sialidase standard stock solution is pressed to the concentration gradient of 5/2.5/1.25/0.625/0.312mU/ml with detection buffer 2
It is diluted to sialidase standard solution, each sialidase standard solution and sample to be tested are sequentially added into detection plate；
(2) fluorescent reagent is diluted with detection buffer 2, is added into the hole of above-mentioned each sialidase standard solution and sample to be tested
It is interior, then will test plate and be placed in 37 DEG C, after being protected from light incubation 1-2 hours, each hole fluorescence intensity is read with microplate reader, according to sialidase
Activity criteria's curve calculates the sialidase activity of sample to be tested, finally obtains sialidase activity/sperm number value i.e.
It can.
7. the application method of the kit of sialidase in a kind of detection external sperm capacitation liquid according to claim 6,
It is characterized by: the detection plate is 96 hole blackboards.
8. the application method of the kit of sialidase in a kind of detection external sperm capacitation liquid according to claim 6,
It is characterized by: being diluted to the concentration of fluorescent reagent with detection buffer 2 is 0.05mM.
9. the application method of the kit of sialidase in a kind of detection external sperm capacitation liquid according to claim 6,
It is characterized by: to read each hole fluorescence in the case where excitation wavelength and launch wavelength are respectively the wavelength of 365nm and 450nm strong for microplate reader
Degree.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610147270.4A CN105779568B (en) | 2016-03-16 | 2016-03-16 | The kit and its application method that sperm quality is assessed after a kind of In-vitro Capacitation |
| PCT/CN2016/081276 WO2017156844A1 (en) | 2016-03-16 | 2016-05-06 | Kit for evaluating sperm quality after in vitro capacitation and method of use thereof |
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| CN201610147270.4A CN105779568B (en) | 2016-03-16 | 2016-03-16 | The kit and its application method that sperm quality is assessed after a kind of In-vitro Capacitation |
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| CN101563134A (en) * | 2006-10-18 | 2009-10-21 | 派利尼斯有限公司 | Method and pharmacological composition for the diagnosis and treatment of male sub-fertility |
| CN103454416A (en) * | 2013-09-22 | 2013-12-18 | 四川大学华西第二医院 | Method for detecting neuraminidase influencing sperm functions |
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| US20040219617A1 (en) * | 2001-02-15 | 2004-11-04 | Sabina Cauci | Enzymatic test for the determination of the risk of pathologies related to the presence of sialidase or prolidase activity in women body fluid samples |
| CN1405562A (en) * | 2002-10-24 | 2003-03-26 | 肖洪武 | Sialidase detection reagent |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101563134A (en) * | 2006-10-18 | 2009-10-21 | 派利尼斯有限公司 | Method and pharmacological composition for the diagnosis and treatment of male sub-fertility |
| CN103454416A (en) * | 2013-09-22 | 2013-12-18 | 四川大学华西第二医院 | Method for detecting neuraminidase influencing sperm functions |
Non-Patent Citations (2)
| Title |
|---|
| Sialidases on Mammalian Sperm Mediate Deciduous Sialylation during Capacitation;Fang Ma et al.;《J Biol Chem》;20121102;第287卷(第45期);实验过程，结果 * |
| 哺乳动物精子质量评定方法研究进展;张明 等;《四川动物》;20070228;第26卷(第1期);第230-233页 * |
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| WO2017156844A1 (en) | 2017-09-21 |
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